Long-term corneal recovery by simultaneous delivery of hPSC-derived corneal endothelial precursors and nicotinamide

Human pluripotent stem cells (hPSCs) hold great promise for the treatment of various human diseases. However, their therapeutic benefits and mechanisms for treating corneal endothelial dysfunction remain undefined. Here, we developed a therapeutic regimen consisting of the combination of hPSC-derived corneal endothelial precursors (CEPs) with nicotinamide (NAM) for effective treatment of corneal endothelial dysfunction. In rabbit and nonhuman primate models, intracameral injection of CEPs and NAM achieved long-term recovery of corneal clarity and thickness, similar with the therapeutic outcome of cultured human corneal endothelial cells (CECs). The transplanted human CEPs exhibited structural and functional integration with host resident CECs. However, the long-term recovery relied on the stimulation of endogenous endothelial regeneration in rabbits, but predominantly on the replacing function of transplanted cells during the 3-year follow-up in nonhuman primates, which resemble human corneal endothelium with limited regenerative capacity. Mechanistically, NAM ensured in vivo proper maturation of transplanted CEPs into functional CECs by preventing premature senescence and endothelial-mesenchymal transition within the TGF-β–enriched aqueous humor. Together, we provide compelling experimental evidence and mechanistic insights of simultaneous delivery of CEPs and NAM as a potential approach for treating corneal endothelial dysfunction.


Introduction
Corneal endothelium maintains corneal hydration and transparency through its barrier and pump function. When the endothelial cell density diminishes to fewer than 500 cells/mm 2 due to dystrophy, trauma, or surgical intervention, corneal endothelial dysfunction will occur and lead to corneal edema, pain, and vision loss, known as bullous keratopathy (1,2). Corneal transplantation represents the major therapeutic approach to treating bullous keratopathy, and it was limited by the global shortage of donor corneas (3,4). Recently, intracameral injection of cultured corneal endothelial cells was reported to improve the visual acuity of patients for 5-year follow-up (5,6). However, the expansion and identification of effector cells for transplantation remains the current challenge.
Stem cell-based therapy holds great promise for the treatment of corneal endothelial dysfunction (7). Previous reports have described the outcomes of corneal endothelial-like cells from somatic stem cells and reprogrammed fibroblasts in animal models (8)(9)(10)(11), but the heterogenous efficiency restricts the development of standard methods for clinical application (12). Based on the pluripotent potential and self-renewal capacity, human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have been used in multiple clinical trials for the treatment of cardiovascular, neurological, and ophthalmic diseases (13). Therefore, hPSCs may provide the promising cell sources to circumvent the severe shortage of donor corneas. Comparatively, autologous hiPSCs avoid the ethical and immune rejection issues associated with hESCs (14,15).
For the induction of corneal endothelial cells from hPSCs, most studies focus on the in vitro morphology and immunostaining methods (16)(17)(18)(19)(20)(21)(22)(23)(24)(25).One group investigated the in vivo therapeutic outcome of hESC-derived cells through the transplantation of tissue-engineered cornea (16,17). More recently, the efficiency of corneal endothelial cells from hESCs and hiPSCs was further evaluated through intracameral injection (26,27). Compared with rapid regeneration of corneal endothelium after injury in rabbit (28), nonhuman primate resembles human corneal endothelium with limited regenerative capacity (29). Therefore, nonhuman primates are more valuable to explore the long-term therapeutic benefits and mechanisms of hPSC-derived cells as preclinical models of treating corneal endothelial dysfunction.
Human pluripotent stem cells (hPSCs) hold great promise for the treatment of various human diseases. However, their therapeutic benefits and mechanisms for treating corneal endothelial dysfunction remain undefined. Here, we developed a therapeutic regimen consisting of the combination of hPSC-derived corneal endothelial precursors (CEPs) with nicotinamide (NAM) for effective treatment of corneal endothelial dysfunction. In rabbit and nonhuman primate models, intracameral injection of CEPs and NAM achieved long-term recovery of corneal clarity and thickness, similar with the therapeutic outcome of cultured human corneal endothelial cells (CECs). The transplanted human CEPs exhibited structural and functional integration with host resident CECs. However, the long-term recovery relied on the stimulation of endogenous endothelial regeneration in rabbits, but predominantly on the replacing function of transplanted cells during the 3-year follow-up in nonhuman primates, which resemble human corneal endothelium with limited regenerative capacity. Mechanistically, NAM ensured in vivo proper maturation of transplanted CEPs into functional CECs by preventing premature senescence and endothelial-mesenchymal transition within the TGF-β-enriched aqueous humor. Together, we provide compelling experimental evidence and mechanistic insights of simultaneous delivery of CEPs and NAM as a potential approach for treating corneal endothelial dysfunction.
Long-term corneal recovery by simultaneous delivery of hPSC-derived corneal endothelial precursors and nicotinamide (CEPs), and corneal endothelial-like cells (CECs) from hESC line H1 ( Figure 1A). The hESCs stained positive for pluripotent markers OCT4 and NANOG (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/ JCI146658DS1). Following 5 days of neural crest induction, the cells exhibited cobblestone morphology with positive stained neural crest markers P75, HNK-1, AP-2α, and AP-2β (Supplemental Figure 1A). Flow cytometry analysis revealed that the purity of P75 + /HNK-1 + cells reached 88.17% ± 0.93% (Supplemental Figure 1B). The NCCs exhibited multipotent capacity of generating peripheral neurons, corneal keratocytes, and mesenchymal stem cells (Supplemental Figure 2). The NCC-derived peripheral neurons stained positive for β-tubulin III and peripherin, and corneal keratocytes stained positive for keratocan, vimentin, and F-actin. The NCC-derived mesenchymal stem cells (MSCs) could differentiate into adipoblasts, osteoblasts, and chondroblasts, which were characterized by Oil In this study, we developed a therapeutic regimen consisting of the combination of hPSC-derived corneal endothelial precursors with nicotinamide for effective treatment of corneal endothelial dysfunction. When intracamerally injected in rabbit and nonhuman primate models, they achieved long-term recovery of corneal clarity and thickness, accompanied by the proper maturation and integration with host resident cells. The transplanted cells survived after 3-year follow-up in the nonhuman primate model. In addition, the long-term therapeutic mechanisms were compared between the rabbit and nonhuman primate models.

Directed differentiation of hESCs into corneal endothelial precursors.
Through the recapitulation of corneal endothelial development, we developed a chemically defined method to induce the differentiation of neural crest cells (NCCs), corneal endothelial precursors cells stained negative for OCT4 and NANOG and had no teratoma formation within 3 months of subcutaneous injection in NOD/ SCID mice (Supplemental Figure 3, B-D). These results indicate that the cells have differentiated from neural crest stage and represent the fate-committed corneal endothelial precursors. In addition, we performed a time-course transcript analysis of the differentiated cells. Following rapid decline of pluripotent genes OCT4 and NANOG, P75 and AP-2α were upregulated after neural crest induction and subsequently downregulated after corneal endothelial differentiation, while ZO-1 and ATP1A1 were continuously elevated ( Figure 1D). Immunostaining showed that the percentage of Ki67-positive cells gradually reduced with the duration of differentiation (Supplemental Figure 1, C and D). Collectively, these data suggest that the inductive differentiation recapitulates the progression of corneal endothelial development.
Stable recovery with simultaneous delivery of hESC-derived CEPs and nicotinamide. Given the role of nicotinamide (NAM) in stem cell differentiation, survival, and EMT inhibition (33-35), we explored whether NAM can improve the therapeutic effect of CEP injection. Therefore, the rabbits were injected with 8 × 10 5 cells containing 50 mM NAM and 100 μM Y27632, and 500 mM NAM and 10 mM Y27632 eye drops were applied 4 times per day ( Figure 3A), with CEP injection, NAM alone without Y27632, and CEP plus NAM without Y27632 as controls. Consistently, all control rabbits showed corneal edematous or slower improvement ( Figure 3, B and C and Supplemental Figure 5). However, when Transient recovery with simple injection of hESC-derived cells and Y27632. To evaluate the therapeutic effects of differentiated cells, rabbit central corneal endothelium (9 mm diameter) was scraped from Descemet's membrane as the model of corneal endothelial dysfunction (Supplemental Figure 4). A quantity of 8 × 10 5 hESC-derived NCCs, CEPs, or CECs supplemented with 100 μM Y27632 was injected into the anterior chamber, with primary cultured human CECs (pCECs) as positive control. The rabbits injected with pCECs gradually resolved corneal edema with the recovery of normal clarity and thickness after 14 days. However, all rabbits injected with hESC-derived cells presented transient recovery and became re-edematous after 14 days, among which the CEPs exhibited better than NCCs and CECs ( Figure 2, A and B). In addition, the rabbits injected with sham operation, medium, or Y27632 alone showed continuous edematous or slower improvement (Supplemental Figure 5).
To explore the cause of corneal re-edema, we performed whole-mounted staining with the human nuclei determinant HuNu and corneal endothelial markers ZO1 and ATP1A1. As shown in Figure 2C, the transplanted CEPs exhibited sporadic and irregular distribution of ZO1 and ATP1A1. As in previous reports cells at day 14, although partial cells exhibited weaker F-actin staining than host endothelium ( Figure 3F and Supplemental Figure 10). By contrast, in rabbit corneas without NAM treatment, both transplanted cells and neighboring host cells displayed the fibroblastic morphology ( Figure 3F), suggesting the inhibitory effect of NAM on EnMT as our previous description (35). When NAM was withdrawn after CEP injection, corneal re-edema appeared in 40% of the rabbits, indicating continuous NAM treatment is essential for the maintenance of corneal recovery (Supplemental Figure 11). Collectively, these results suggest that short-term recovery is achieved through the contribution of transplanted CEPs and NAM, but that longterm recovery relies on the stimulation of resident endothelial regeneration with the loss of transplanted cells.
NAM orchestrates in vivo functional maturation of hESC-derived CEPs. To explore the potential mechanism of NAM treatment, we collected the corneal endothelium within the scraped area and compared the expression patterns of transplanted cells using human-specific primers. The expressions of neural crest genes decreased gradually, while corneal endothelial genes increased after transplantation ( Figure 4A and Supplemental Figure 12A). The transplanted cells stained positive for neural crest marker P75 and discontinuous corneal endothelial markers ZO-1, ATP1A1, combined with NAM, CEP injection rapidly improved corneal clarity and restored normal thickness at day 7 ( Figure 3, B and C). Confocal microscopy observation showed that the rabbit corneas possessed intact endothelium with relative regular morphology and favorable endothelial cell density (Figure 3, D and E), but the transplanted cells didn't achieve uniform alignment as host resident cells (Supplemental Figure 6). The recovery was continuously maintained during the follow-up to 8 weeks (Supplemental Figure  7A). No significant changes of intraocular pressure were observed (Supplemental Figure 8). The human specific Actin was detected only in the corneal endothelium and trabecular tissue within the first 4 weeks (Supplemental Figure 9).
To evaluate the survival and integration of transplanted cells, we performed double immunofluorescence staining with the human nuclei determinant HuNu and cell-surface determinant TRA-1-85, combined with phalloidin to label cytoskeleton F-actin. The transplanted cells covered the scraped area within 2 weeks, decreased after 4 weeks, and were completely lost after 8 weeks. The proliferating cell nuclear antigen-positive (PCNA-positive) cells were detected among the host resident corneal endothelium (Supplemental Figure 7). More specifically, the transplanted CEPs with NAM treatment formed a regularly aligned monolayer and integrated with host and SLC4A11 at day 7 (Supplemental Figure 12B). After 14 days of transplantation with NAM treatment, they stained negative for P75 and AP-2β, and displayed an intact positive staining pattern for corneal endothelial markers, while the cells without NAM still exhibited sparse and irregular staining ( Figure 4B). Furthermore, NAM treatment reduced the mRNA transcripts of EnMT-and senescence-associated genes ( Figure 4C). The transplanted cells exhibited negative staining of α-SMA and fibronectin as well as the reduced senescent cells ( Figure 4D).
Previous studies have reported the involvement of TGF-β signaling in the senescence and EnMT of corneal endothelial cells (36)(37). Therefore, we collected the rabbit aqueous humor and found increased TGF-β1 levels after endothelial scraping (Supplemental Figure 13). The inhibition of NAM on TGF-β1-induced EnMT and cellular senescence was confirmed by the in vitro cultured hESC-derived CEPs (Supplemental Figure 14A). RNA microarray analysis showed significant changes with NAM treatment (Supplemental Figure 14B). The core member in TGF-β signaling pathway, TGFB2, 2 classical EnMT related genes, ACTA2 (α-SMA) and FN1 (fibronectin), and 2 cellular senescence-associated genes, including P16 and P21, declined with NAM treatment, while the corneal endothelial gene COL8A2 and several anti-EnMT genes were elevated (38)(39)(40). KEGG analysis revealed that differentially expressed genes were involved the TGF-β signaling pathway, cellular senescence, and focal adhesion (Supplemental Figure 14C). Further analysis and qPCR validation revealed that NAM repressed TGF-β and cellular senescence-related gene sets, and induced the upregulation of focal adhesion-associated genes (Supplemental Figure 14, D and E). Overall, these results suggest that NAM orchestrates in vivo functional maturation of hESCderived CEPs through multiple mechanisms, similar to previous descriptions (33, 34, 41). To further corroborate the therapeutic strategy, we repeated the inductive method and intracameral injection with 2 human iPSC lines DYR0100 and U2. The transplantation of hiPSC-derived CEPs and NAM resolved corneal edema with the recovery of corneal clarity and thickness in rabbits. The transplanted cells displayed regularly arranged staining of corneal endothelial markers (Supplemental Figure 15).
Long-term outcomes of hESC-derived CEP injection and NAM in primate models. Considering the limitation of the rabbit model (28), a nonhuman primate model was used to further elucidate the therapeutic benefits of hESC-derived CEP injection and NAM treatment. Central corneal endothelium (6 mm diameter) of 5 cynomolgus monkeys was scraped to generate the preclinical model. One monkey (M1) was injected with 6 × 10 5 CEPs, 4 monkeys (M2-M5) were injected with CEPs and NAM treatment. During the follow-up of 3 and 6 months, M1 showed persistent corneal edema, while M2-M5 resolved corneal edema with recovered corneal clarity and thickness within 2-8 weeks, although the initial surgical operation caused iris distortion in M4 ( Figure 5, A and  B). Confocal microscopy confirmed that the corneal endothelium (M2 and M3 for 3 months, M4 and M5 for 6 months) displayed a relative regular morphology with the density of 2362 ± 108.25 cell/ mm 2 (M2), 2887 ± 54.49 cell/mm 2 (M3), 2600 ± 154.11 cell/mm 2 (M4), and 2050 ± 127.48 cell/mm 2 (M5) ( Figure 5C). B-mode ultrasound, fundus photography, and intraocular pressure confirmed no abnormal pathological changes (Supplemental Figure 16).
To verify the long-term outcome, we followed the M4 up to 36 months. Transplanted cells were not detected in the iris, trabecular, and internal organs as confirmed by PCR (Supplemental Figure 17A). The recovered corneal clarity and thickness were maintained with more regular endothelial morphology and cell density of 2431 ± 152.45 cell/mm 2 , which was greater than 70% of the normal eye ( Figure 5, A-C). To evaluate the survival of transplanted cells, we screened several human antibodies, including HuNu, TRA-1-85, MTCO2, and SC101 (Supplemental Figure  18), and identified the human-specific antibody SC-121 to discriminate the transplanted cells from monkey resident cells. As shown in Figure 5D, the transplanted cells were detected within the complete central area of 2 mm diameter and partial middle area of 2-4 mm diameter. However, they were not detected in the peripheral area of 4-6 mm diameter, which was covered with only monkey cells. The PCNA-positive cells were also detected in the peripheral area after CEP transplantation and NAM treatment (Supplemental Figure 17B). Moreover, transplanted cells exhibited regular hexagonal morphology and corneal endothelial marker expression pattern, similar to the normal corneal endothelium ( Figure 5E). These results suggest that the strategy of CEP transplantation and NAM treatment achieves the long-term recovery of corneal clarity and thickness in nonhuman primates. Although it stimulates the partial regeneration of resident monkey cells, major therapeutic effects predominantly rely on the replacing function of transplanted human cells.

Discussion
hPSCs provide a promising alternative cell source for treating corneal endothelial dysfunction. Although several studies have reported that corneal endothelial differentiation is induced from hPSCs, the long-term therapeutic benefits and mechanisms remain unclear, especially in a nonhuman primate model with similar limited regenerative capacity to human corneal endothelium. In the present study, we generated the fate-committed corneal endothelial precursors from 1 hESC line and 2 hiPSC lines. When combined with NAM treatment, intracameral injection of hPSC-derived CEPs achieved long-term effective treatment of corneal endothelial dysfunction in rabbit and nonhuman primate models. Mechanistically, NAM ensured in vivo proper maturation of transplanted CEPs into functional CECs by preventing their EnMT and premature senescence.
The corneal endothelium is derived from cranial neural crest cells, which migrate and direct into corneal endothelial fate under the local microenvironment (42,43). Similar to previous reports (18,20,21), we developed a 2-step method to induce the differentiation of neural crest cells and corneal endothelial-like cells from hESCs. However, when injected in rabbit model, the corneal endothelial-like cells showed almost no improvement, while the neural crest cells achieved transient corneal recovery. Therefore, we identified the middle-stage corneal endothelial precursors. Benefiting from the high purity of neural crest cells differentiated from hESCs, the CEP population was relative homogenous according to immunostaining and FACS analysis. Moreover, CEPs express neural crest markers P75 and AP-2β and corneal endothelial markers ZO-1, ATP1A1, and AQP1. Meanwhile, they have lost HNK-1 expression and are negative for mature corneal endothelial markers SLC4A11 and N-cadherin. The above evidence suggests that the CEP population represents putative fate-committed corneal endothelial precursors from neural crest cells.
The ROCK inhibitor Y27632 has been used in the intracameral injection of human CECs to promote their attachment and survival (44). Accordingly, we injected hESC-derived CEPs with Y27632 in rabbit models and found better improvement than NCCs and CECs within 3 days. However, the corneas became re-edematous because of TGF-β-induced EnMT of the transplanted cells. These data indicate that Y27632 alone is insufficient to maintain the in vivo long-term outcome of hESC-derived CEPs. Therefore, we selected NAM as another adjunctive supplement for transplantation. In line with a previous report (35), NAM alone promoted rabbit corneal endothelial wound healing, but the recovery of corneal clarity and thickness was slower and incomplete within 14 days. Comparatively, the CEP transplantation and NAM treatment achieved a rapid and complete recovery within 7 days, accompanied by a structural and functional integration with host cells. It should be mentioned that partial cells showed weaker F-actin than host resident cells, indicating that they were still immature after 14 days of transplantation in rabbits, even when normal corneal clarity and thickness restored completely.
Previous studies have described that stem/progenitor cell transplantation may promote the regeneration of host resident cells (45,46). To demonstrate the therapeutic mechanisms of CEP transplantation and NAM treatment, we compared the short-term and long-term fate decision of transplanted cells in rabbits and nonhuman primates. In rabbits with rapid endothelial regenerative capacity, the strategy of CEP transplantation and NAM treatment achieved persisted corneal recovery from 7 days to 8 weeks. However, the transplanted cells were completely lost at 8 weeks, It should be mentioned that there are still some limitations for the clinical translation of CEP transplantation and NAM treatment, such as complete transparency recovery, stromal haze, and irregular endothelial morphology. The initial intraocular inflammation represents a major problem for the success of corneal recovery in nonhuman primate models. Although the moderate-to-severe inflammatory response was found in 1 monkey, once controlled rapidly, no further inflammation and rejection occurred during the 3-year follow-up.
In summary, this study described a therapeutic regimen consisting of the combination of hPSC-derived CEPs and nicotinamide for the treatment of corneal endothelial dysfunction. Nicotinamide promoted the proper maturation of transplanted CEPs into functional CECs by preventing EnMT and premature senescence within the TGF-β-enriched aqueous humor. Long-term corneal recovery relied on the replacing function of transplanted cells in nonhuman primate model.

Animals.
One-year-old male New Zealand white rabbits (Kangda Biotechnology) and 3-to 5-year-old female cynomolgus monkeys (Guidong Quadrumana Development & Laboratory Co.) were used to establish the model of the corneal endothelial dysfunction for transplantation experiment. Six-to eight-week-old male NOD/SCID mice (Vital River Laboratory Animal Technology Co.) were used for the teratoma formation experiment.
Cell lines. The human ES cell line H1 was provided by Zhengqin Yin. The human iPS cell line DYR0100 and U2 were provided by the Stem Cell Bank, Chinese Academy of Sciences and Beijing Cellapy Biotechnology Company. The hESCs and hiPSCs were cultured in serum-free medium mTeSR1 (STEMCELL Technology) in plates coated with growth factor-reduced Matrigel (BD Biosciences). The hESCs and hiPSCs were manually passaged once every 4 to 5 days with Accutase (Sigma). All cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . The medium was changed every day.
Peripheral neuron, mesenchymal cell, and corneal keratocyte differentiation. For peripheral neuron differentiation, NCCs were cultured accompanied the presence of dividing cells in resident corneal endothelium. This evidence supports the theory that short-term corneal recovery in rabbits is achieved through the replacing function of transplanted cells, while long-term recovery relies on the stimulated regeneration of resident cells. In nonhuman primate models with limited endothelial regenerative capacity, the strategy of CEP transplantation and NAM treatment also achieved persisted corneal recovery from 8 weeks to 36 months. When checking the survival of transplanted cells at 36 months, we found the peripheral scraped area was covered with only monkey cells, the middle area was mixed with both monkey and transplanted human cells, and the central area was covered with only transplanted human cells. These findings support that the transplanted cells actually stimulate the limited regeneration of monkey resident cells, similar to a previous description of descemetorhexis without endothelial keratoplasty in human (47)(48)(49). However, the long-term corneal recovery predominantly relies on the replacing function of transplanted human cells, which was different from the dependence of stimulated endothelial regeneration in rabbits. labeled with Cyanine-3-CTP. The labeled cRNAs were hybridized onto the microarray. After washing, the arrays were scanned by the Agilent Scanner G2505C (Agilent Technologies). Feature Extraction software (version 10.7.1.1, Agilent Technologies) was used to analyze array images to get raw data. Next, the raw data was normalized with the quantile algorithm. The probes detected with 3 samples in any group were chosen for further data analysis. Differentially expressed genes were then identified through fold change as well as P value calculated with t test. The threshold set for up-and downregulated genes was log2 fold change greater than 0.5 and P value less than 0.05. Afterwards, KEGG analysis was applied to determine the roles of these differentially expressed mRNAs. The microarray data have been stored in the Gene Expression Omnibus (GEO). Data can be accessed through http://www.ncbi.nlm.nih.gov/ geo (accession number: GSE183786).
Statistics. The Statistical Package for the Social Sciences version 17.0 software and Prism 8 (Graphpad) software were used for statistical analysis. Comparison between 2 experimental groups was determined with 2-tailed Student's t test. More than 2 groups were made using ANOVA followed by Tukey's honestly significant difference (HSD) test. Data in this study represent at least 3 independent experiments and are presented as mean ± SEM. Results were considered significant at *P < 0.05 and **P < 0.01, and specific comparisons are indicated in the respective figure legends.
Study approval. All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and they were approved by the Ethics Committee of Shandong Eye Institute (accreditation no. 2012-G-D01).

Author contributions
WS and QZ conceived and designed the project, interpreted results, and wrote the manuscript. ZL performed experiments, analyzed data, and wrote the manuscript. HD performed cell experiments and immunostaining experiments. WL and BM performed cell experiments. YJ, CZ, XW, YG, and CD performed animal experiments. SD and BZ analyzed data. DL performed animal examination. LX and YC revised the manuscript. ZL is listed first in the order of co-first authors because of his major contributions to research design and manuscript preparation.